Development of pancreas storage solutions: Initial screening of cytoprotective supplements for β-cell survival and metabolic status after hypothermic storage

Abstract

Insulin-dependent diabetes mellitus is one of the leading causes of death world-wide. Donor-derived pancreas and Islet of Langerhans transplantation are potential cures; however, postmortem ischemia impacts islet quality. The murine βt3 cell line was employed as a model to study cell viability and proliferation after hypothermic storage by comparing Belzer's Machine Perfusion Solution with Unisol™ Solution. The objective was to determine which of these solutions provided the best base line support for βt3 cells and to screen potential cytoprotective additives to the solutions. Initial βt3 cell viability was similar in the two storage solutions; however, better proliferation was observed after storage in Unisol Solution. The caspase inhibitor, Q-VD-OPH, and α-tocopherol improved viability in both storage solutions, suggesting that apoptotic pathways may be responsible for cell death during hypothermic storage of βt3 cells. Analysis of apoptosis markers, caspase activity, and DNA laddering showed a reduction in apoptosis when these additives were included. The effects of Q-VD-OPH and α-tocopherol were also synergistic when employed together during either hypothermic exposure, post-hypothermic physiologic incubation, or combinations of hypothermic exposure and physiologic incubation. These results suggest that both supplements should be included in pancreas hypothermic storage solutions and in islet culture media during post-isolation culture prior to transplantation.

FIG. 1.

Cell viability of βt3 cells after storage at 4°C. βt3 cells were stored for the indicated time periods in either Unisol™ or BMPS. Then cell viability was measured every day for 7 days post-storage. Values represent the mean (±SEM) for 24 replicates. Differences in viability were considered significant by one way ANOVA, p<0.05.

FIG. 2.

Cell viability after storage with an apoptotic inhibitor. βt3 cells were stored for 16–24 hours at 4°C in BMPS (A) or Unisol™ (B) with the apoptotic inhibitor Q-VD-OPH (QVD) at the indicated concentrations. Cell viability was measured immediately after storage and for several days post-storage as indicated. Values represent the mean (±SEM) for 9 replicates. Differences in viability were considered significant by one-way ANOVA.

FIG. 3.

Cell viability after storage with the antioxidant, α-tocopherol (αT). βt3 cells were stored for 16–24 hours at 4°C in BMPS (A) or Unisol (B) with the indicated concentrations of α-tocopherol. Cell viability was measure immediately after storage and for several days post-storage as indicated. Values represent the mean (±SEM) for 6 replicates. Differences in viability were considered significant by one-way ANOVA.

FIG. 4.

Cell viability after storage at 4°C with α-tocopherol and QVD. βt3 cells were stored for 16–24 h at 4°C in BMPS (A) or Unisol™ (B) alone, with 50 μM α-tocopherol, 10 μM QVD, or a combination of both additives. Cell viability was measured immediately after storage and for several days post-storage as indicated. Values represent the mean (±SEM) for 24 replicates. Differences in viability were considered significant by one-way ANOVA.

FIG. 5.

Cell viability after storage and recovery with α-tocopherol and QVD. βt3 cells were stored for 16–24 h at 4°C in BMPS (A) or Unisol (B) alone, with 50 μM α-tocopherol, 10 μM QVD, or a combination of both additives. Additives were added during storage at 4°C, during recovery at 37°C, or both. Cell viability was measured immediately after storage and for several days post-storage as indicated. Values represent the mean (±SEM) for 21 replicates. Differences in viability were considered significant by one-way ANOVA.

FIG. 6.

Apoptotic activity after storage with α-tocopherol and QVD. βt3 cells were stored for 16–24 h at 4°C in BMPS alone, with 50 μM α-tocopherol, 10 μM QVD, or a combination of both additives. Apoptotic activity was measured by nexin staining, caspase activity, and the detection of DNA laddering (TUNEL). Apoptotic cells are expressed as the percentage of cells positive for apoptosis markers out of 1000 counted cells. Values represent the mean (±SEM) for 12 replicates. Differences in viability were considered significant by one-way ANOVA.